Our previous work demonstrated that Selective propagation of functional mtDNA during oogenesis restricts the transmission of a deleterious mtDNA mutation (Hill et al, Nature Genetics 2014,). To understand the cellular processes that may influence mtDNA selection and transmission, we carried out RNAi screening in both drosophila ovary and cultured cells for genes required for mtDNA replication. We identified a mitochondrial outer membrane protein, MDI that was essential for mtDNA replication in ovary. MDI recruits Larp, a translation-stimulating protein to the mitochondrial outer membrane. MDI-Larp complex promote the synthesis of a subset of nuclear-encoded mitochondria proteins by cytosolic ribosomes on mitochondrial surface. Proteomic study reveals that MDI-Larp targets are key components in mtDNA replication and mitochondrial biogenesis. Lack of MDI abolishes mtDNA replication in the ovary. The eggs of mdi mutant flies are largely devoid of mtDNA and arrest at embryonic development. In addition, mdi mutant females fail to limit the transmission of a deleterious mtDNA mutation to their progeny. Our work illustrates the protein synthesis by cytosolic ribosomes on mitochondrial surface, and its essential role in mitochondrial biogenesis and mtDNA replication. Our work also demonstrates how this novel translational regulation impacts mtDNA inheritance. MDI acts in two key aspects of mtDNA inheritance: limiting the transmission of harmful mtDNA mutations, and providing enough mitochondria/mtDNA for early embryogenesis. We identified a mitochondrial cyclic nucleotide phosphodiesterase, prune was essential for mtDNA maintenance in cultured cells. Disruption of prune elevates cAMP level inside the matrix, which subsequently down- regulates TFAM and mtDNA level through PKA. Our work proves the prevalence of mitochondrial cAMP signaling in metazoan. More importantly, it demonstrates an expanded role of mitochondrial cAMP signaling in regulating mitochondrial biogenesis. To advance our understanding of the pathogenic processes underlying mtDNA diseases and to test potential therapeutic interventions, we have pursued biochemical and genetic characterization on a previously isolated conditional lethal mtDNA mutation, mt:CoIT300I. We found that the decrease in cytochrome c oxidase (COX) activity in mt:CoIT300I was ascribed to the destabilization of cytochrome a heme. Consistently, the viability of homoplasmic flies was fully restored by expressing an alternative oxidase, which specifically bypasses the cytochrome chains. We developed a genetic scheme to induce tissue-specific homoplasmy in heteroplasmic flies. We found that homoplasmy in the nerve system caused severe neurodegeneration. The degeneration was suppressed by improving mitochondrial Ca2+ uptake, suggesting that Ca2+ mishandling contributes to the pathogenesis of mtDNA disorders. We also carried out genetic analysis on a mitochondrial i-AAA protease in Drosophila. We found that i-AAA was essential for removing unfolded proteins and maintaining mitochondrial membrane architecture. Loss of i-AAA led to the accumulation of oxidative damage, progressive deterioration of membrane integrity and apoptotic neural and muscular degeneration. Containing ROS level suppressed the degenerative phenotypes, suggesting it could be a potential strategy to manage mitochondrial proteostasis deficiency. Currently we are testing whether MDI-Larp complex might function as a sensor for mitochondrial activity to promote mtDNA replication in healthy mitochondria. We are also investigating how mitochondrial nucleoid proteins (mtDNA associated proteins) influence mtDNA packing, movement and segregation, and how these processes impact mtDNA transmission and selection in female germ line.